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  • Transplanted sheets of human retina and retinal pigment epithelium develop normally in nude rats. 12137757

    This study investigated whether transplanted sheets of human fetal retina together with its retinal pigment epithelium (RPE) could develop and maintain their cytoarchitecture after long survival times. Transplant recipients were nine albino athymic nu/nu rats with a normal retina. The donor tissue was dissected from fetuses of 12-17 weeks gestational age. Transplants were analyzed at 5-12 months after surgery by light and electron microscopy, and immunohistochemistry with various antibodies specific for rhodopsin, S-antigen, transducin, neurofilament and synaptophysin. In 4 of 11 transplants, the RPE stayed as a monolayer sheet and supported the development of the retinal sheet with a normal lamination, including photoreceptor inner and outer segments. Cones and rods in the organized transplants were labeled with different photoreceptor markers. Inner and outer plexiform layers, containing cone pedicles and rods spherules, were immunoreactive for synaptophysin. As the recipients had a normal retina, transplant/host integration was not expected. However, at the transplant/host interface, there were sometimes areas without glial barriers, and neurofilament-containing processes could be observed crossing between transplant and host. In other, more disorganized transplants, the RPE cells were partially dispersed or clumped together in clusters. Such transplants developed photoreceptors in rosettes, often with inner and outer segments.In conclusion, sheets of human fetal retina transplanted together with its RPE to the subretinal space of nude rats can develop and maintain perfectly laminated transplants after long survival times, indicating the potential of applying cotransplantation to human patients with retinal diseases.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
  • Carrier-free epithelial cell sheets prepared by enzymatic degradation of collagen gel. 20603893

    Limbal stem-cell deficiency by ocular trauma or disease causes corneal opacification and vision loss. Conventional tissue engineering using biodegradable scaffolds has met with limited success. In this study, we developed a novel method for preparing carrier-free epithelial cell sheets, which have potential for use in repairing defects of the ocular surface. Stratified corneal epithelial cell sheets were prepared in culture dishes coated with biodegradable type I collagen. Haematoxylin and eosin staining, electron microscopy and immunohistochemistry were performed to characterize the cell sheets. Then, carrier-free epithelial sheets were successfully engineered using specific collagenase to degrade the collagen gel. Cell sheets of four to six cell layers after culture for 14 days were similar to natural rabbit corneal epithelia, as shown by pathological examination. Microvillus, tight cell-cell junctions and desmosome junctions were observed via electron microscopy. K3 and basement membrane components, such as type IV collagen and laminin, were expressed in the cells sheets and integrin β1 was maintained in basal cells. This novel method of using collagenase to degrade collagen gel is both simple and effective in preparing intact carrier-free epithelial cell sheets. Such sheets have great potential for application during in vivo corneal regeneration.
    Document Type:
    Reference
    Product Catalog Number:
    CBL218
    Product Catalog Name:
    Anti-Keratin K3/K76 Antibody, clone AE5
  • Tooth root regeneration using dental follicle cell sheets in combination with a dentin matrix - based scaffold. 22192537

    Stem cell mediated tissue engineering has been acknowledged as a prospective strategy for repairing and replacing damaged and lost tissues. However, the low survival rate of implanted stem cells proves to be a major challenge in the management of transplantation failures. While previous studies have indicated the effectiveness of tissue engineered cell sheets in improving the survival rate of implanted cells, we have recently demonstrated the use of treated dentin matrix (TDM) as a biological scaffold and dental follicle cells (DFCs) as the seeding cells for dentinogenesis and tooth root construction. This study proposes a strategy utilizing TDM with human dental follicle cell sheets (DFCSs) for root regeneration. The biological characteristics and changes of human DFCSs under the effect of TDM were studied with scanning electron microscopy, transmission electron microscopy, immunofluorescence microscopy, immunohistochemistry and quantitative real-time PCR. DFCSs combined with TDM were implanted subcutaneously into the dorsum of mice. Histological examination of the harvested grafts revealed a whirlpool-like alignment of the DFCs in multiple layers that were positive for COLI, integrinβ1, fibronectin and alkaline phosphatase (ALP), suggestive of the formation of a rich extracellular matrix. DFCSs, under the effect of TDM, highly expressed DMP-1 and bone sialoprotein (BSP), indicating their potential for odontogenesis and osteogenesis. Importantly, in vivo, TDM could induce and support DFCSs to develop new dentin-pulp like tissues and cementum-periodontal complexes that were positive for markers such as DSP, nestin and VIII factors, COLI and cementum attachment protein (CAP), implying successful root regeneration. Therefore, DFCSs combined with TDM may prove to be a better strategy for the construction of tooth root, and is a prospective approach that could be utilized for the treatment of root or tooth defect or loss in future.
    Document Type:
    Reference
    Product Catalog Number:
    AP192F
    Product Catalog Name:
    Donkey Anti-Mouse IgG Antibody, FITC conjugate, Species Adsorbed
  • Heat shock attenuates VEGF expression in three-dimensional myoblast sheets deteriorating therapeutic efficacy in heart failure. 22129892

    Myoblast sheet transplantation is a promising novel treatment for ischemic heart failure. The aim of this study was to test the hypothesis that heat shock (HS) pre-treatment affects the angiogenic properties of myoblast sheets in vivo and in vitro.We studied HS preconditioning of L6 myoblast sheets in relation to their apoptosis, proliferation, and vascular endothelial growth factor (VEGF)-associated responses under normoxia and under hypoxia in vitro. In vivo evaluation of their therapeutic effect was performed with 60 male Wistar rats divided into 3 groups (20 each): sole left anterior descending (LAD) ligation (control); LAD ligation and non-conditioned sheet transplantation (L6 No-Shock); and LAD ligation and L6-heat shock conditioned sheet transplantation (L6 Heat-Shock). Left ventricular function was evaluated by echocardiography after 3, 10, and 28 days.Expression of HSP70/72 was strongly induced 24 hours after HS, and thereafter it decreased notably during 72 hours in hypoxia. Under normal growth conditions, HSP70/72 expression remained stable. HS delayed apoptosis-associated caspase-3 expression during 24-hour hypoxia compared to non-treated controls. However, VEGF expression reduced significantly in the heat shock pretreated sheets. Ejection fraction of the L6-myoblast HS pre-treatment group (L6 Heat-Shock) decreased gradually during follow-up, in the same pattern as the controls. However, these functional parameters improved in the L6-myoblast normal sheet group (L6 No-Shock) at the tenth day and remained significantly better.HS protects myoblast sheets from hypoxia-associated apoptosis in vitro, but reduces VEGF expression of the sheet, leading to lower therapeutic effect in heart failure.
    Document Type:
    Reference
    Product Catalog Number:
    AB7356
    Product Catalog Name:
    Anti-von Willebrand Factor Antibody
  • Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. 388439

    A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
  • The use of human mesenchymal stem cell-derived feeder cells for the cultivation of transplantable epithelial sheets. 19136703

    PURPOSE: To report the efficacy of human bone marrow-derived mesenchymal stem cells as a source of feeder cells for the cultivation of transplantable corneal epithelial cell sheets. METHODS: Human mesenchymal stem cells (marrow adherent stem cells; MASCs) were cultured in alpha-modified Eagle's medium with 10% serum and were treated with mitomycin C. Expression of cytokines in MASCs was confirmed by reverse transcription-polymerase chain reaction. Human limbal epithelial cells were cocultured with MASCs or 3T3 feeder cells to compare colony-forming efficiency (CFE). Limbal epithelial cells were cultured on MASCs or 3T3 feeder cells at the air-liquid interface to allow stratification, and stratified epithelial sheets were analyzed by immunohistochemistry against cytokeratin 3 (K3), K15, p63alpha, and ABCG2. Rabbit limbal epithelial cell sheets were cultivated with MASC feeder cells and transplanted to the ocular surface of the limbal-deficient rabbits. Epithelial grafts were observed by slit lamp microscopy for 4 weeks and then evaluated by histology and immunohistochemistry against K3 and K4. RESULTS: MASC feeder cells expressed keratinocyte growth factor, hepatocyte growth factor, and N-cadherin. The CFE of human limbal epithelial cells was similar in MASC and 3T3 feeder groups. Stratified cell sheets were successfully cultivated with MASC feeder cells expressing K3, K15, p63alpha, and ABCG2. Transplanted epithelial sheets regenerated the corneal phenotype in limbal-deficient rabbits. CONCLUSIONS: MASC-derived feeder cells are suitable for the engineering of epithelial sheets, avoiding the use of potentially hazardous xenologic feeder cells.
    Document Type:
    Reference
    Product Catalog Number:
    MAB4146
  • Conversion of alpha-helices into beta-sheets features in the formation of the scrapie prion proteins. 7902575

    Prions are composed largely, if not entirely, of prion protein (PrPSc in the case of scrapie). Although the formation of PrPSc from the cellular prion protein (PrPC) is a post-translational process, no candidate chemical modification was identified, suggesting that a conformational change features in PrPSc synthesis. To assess this possibility, we purified both PrPC and PrPSc by using nondenaturing procedures and determined the secondary structure of each. Fourier-transform infrared (FTIR) spectroscopy demonstrated that PrPC has a high alpha-helix content (42%) and no beta-sheet (3%), findings that were confirmed by circular dichroism measurements. In contrast, the beta-sheet content of PrPSc was 43% and the alpha-helix 30% as measured by FTIR. As determined in earlier studies, N-terminally truncated PrPSc derived by limited proteolysis, designated PrP 27-30, has an even higher beta-sheet content (54%) and a lower alpha-helix content (21%). Neither PrPC nor PrPSc formed aggregates detectable by electron microscopy, while PrP 27-30 polymerized into rod-shaped amyloids. While the foregoing findings argue that the conversion of alpha-helices into beta-sheets underlies the formation of PrPSc, we cannot eliminate the possibility that an undetected chemical modification of a small fraction of PrPSc initiates this process. Since PrPSc seems to be the only component of the "infectious" prion particle, it is likely that this conformational transition is a fundamental event in the propagation of prions.
    Document Type:
    Reference
    Product Catalog Number:
    AG210
    Product Catalog Name:
    Prion Protein, recombinant